The uroepithelium, which lines the inner surface of the bladder, forms an impermeable barrier and also functions as an integral part of a sensory web. Through uroepithelial-associated channels and receptors, the uroepithelium receives sensory input such as changes in hydrostatic pressure and binding of mediators such as ATP. These input signals stimulate membrane turnover in the outermost umbrella cell layer and release of sensory output from the uroepithelium in the form of mediators that communicate changes in the uroepithelial milieu to the underlying tissues, altering the function of the bladder. Adenosine is a universally produced nucleoside that participates in the normal function of all organ systems, and preliminary data indicates that the uroepithelium is a site of adenosine biosynthesis and expresses all four adenosine receptors (A1, A2a, A2b, and A3). Adenosine binding to these receptors stimulates exocytosis in the umbrella cell layer. The goal of this proposal is to test the hypothesis that adenosine, acting through uroepithelial-associated adenosine receptors, functions as a sensory input molecule that stimulates membrane turnover in the umbrella cell layer and alters bladder function. The first aim of this proposal will define the mechanism and function of adenosine biosynthesis and turnover by the uroepithelium. Selective inhibitors of enzymes/transporters as well as knockout mice lacking expression of adenosine kinase and adenosine deaminase will be used to define the mechanisms of adenosine production and turnover by the uroepithelium. Adenosine and its metabolites will be measured with a novel, highly sensitive and specific 3D-ion trap HPLC-mass spectrometric method. The second aim will define if uroepithelial-associated adenosine receptors modulate bladder function. The Cre/loxP system will be used to generate mice lacking uroepithelial expression of A1 and A2a receptors. Bladder function will be studied in these mice as well as in knockout mice globally lacking A1, A2a, A2b, and A3 receptor expression in all tissues. The third aim will define the role of adenosine and adenosine receptors in modulating membrane turnover in umbrella cells. Selective agonists and knockout mice lacking uroepithelial or global expression of A1 and A2a receptors will be used to define if these receptors are important for regulating endocytosis/exocytosis in the uroepithelium. The fourth aim will explore which signaling pathways act downstream of adenosine receptors to modulate membrane turnover in the uroepithelium. A combined biochemical/pharmacological approach and permeabilized cell systems will be used to define which G proteins and second messenger pathways are involved in regulating membrane traffic in the umbrella cell layer. The results of these studies will increase our understanding of the role of adenosine and its receptors in the uroepithelial-associated sensory web, and will ultimately allow us to understand how perturbations in uroepithelial-associated receptor expression and signal output can contribute to bladder diseases such as interstitial cystitis and detrusor overactivity.Project Narrative. Adenosine is a natural product of cells that regulates the function of the cardiovascular, neuronal, and renal organ systems. This grant will explore if adenosine regulates the delivery and recovery of membrane at the free surface of the cells lining the bladder, and whether adenosine affects bladder filling and voiding. Understanding adenosine function is important because it will help us to understand how the cells lining the bladder can aid in regulating the function of the bladder and how to treat diseases like interstitial cystitis and bladder overactivity.